Protein engineering of a disulfide bond in a beta/alpha-barrel protein.


Abstract

A disulfide bond has been introduced in the beta/alpha-barrel enzyme N-(5'-phosphoribosyl)anthranilate isomerase from Saccharomyces cerevisiae. The design of this disulfide bond was based on a model structure of this enzyme, built from the high-resolution crystal structure of the N-(5'-phosphoribosyl)anthranilate isomerase domain from Escherichia coli. The disulfide cross-link is spontaneously formed in vitro between residues 27 and 212, located in the structurally adjacent alpha-helices 1 and 8 of the outer helical ring of the beta/alpha-barrel. It creates a loop of 184 residues that account for 83% of the sequence of this enzyme, thus forming a quasi circular protein. The cross-linked mutant enzyme displays wild-type steady-state kinetic parameters. Measurements of the equilibrium constant for the reduction of this disulfide bond by 1,4-dithiothreitol show that its bond strength is comparable to that of other engineered protein disulfide bonds. The oxidized, cross-linked N-(5'-phosphoribosyl)anthranilate isomerase mutant is about 1.0 kcal/mol more stable than the wild-type enzyme, as estimated from its equilibrium unfolding transitions by guanidine hydrochloride. Study holds ProTherm entries: 4215, 4216 Extra Details: additive : EDTA(1 mM), disulfide bond; beta/alpha-barrel; quasi circular protein;,engineered protein

Submission Details

ID: XcvfwRwC3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:24 p.m.

Version: 1

Publication Details
Eder J;Wilmanns M,Biochemistry (1992) Protein engineering of a disulfide bond in a beta/alpha-barrel protein. PMID:1581299
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 N-(5'-phosphoribosyl)anthranilate isomerase P00912 TRPF_YEAST